Grasses expressing AtH1 exhibit delayed heading and reduced inflorescenses

ABSTRACT

Novel grass plants, their progeny, and parts thereof are disclosed which have been genetically modified by introduction of a nucleic acid molecule encoding the transcription factor AtH1. This modification causes a heritable change in one or more plant characteristics such as, for example, inhibition of flowering, absence of inflorescence, increased production of tillers, delayed heading, and inhibition of the developmental switch from vegetative to generative growth.

FIELD OF THE INVENTION

The invention relates to grass plants, their progeny, and parts thereof,which have been genetically modified. This modification causes aheritable change in one or more plant characteristics such as, forexample, inhibition of flowering, absence of inflorescence, increasedproduction of tillers, delayed heading, and inhibition of thedevelopmental switch from vegetative to generative growth.

BACKGROUND OF THE INVENTION

The prior art includes grasses that have been mutated such thatflowering and production of inflorescences do not occur. These grasses,however, exhibit other unwanted characteristics such as dwarfism, leafdiscoloration, root failure, and the like. The phrase “geneticallymodified” as used herein does not include chemical or irradiationmutagenesis, nor standard hybridization techniques that produce sterileprogeny. For example, transformation with a nucleic acid to produce analteration in the plant's genetic material is within the scope of theinvention.

The prior art also includes grasses that have been treated withchemicals or phytohormones to inhibit flowering and production ofinflorescences. But genetic modification in accordance with the presentinvention results in a change in heritable traits and does not requiresuch treatment. Change in one or more characteristics of a geneticallymodified grass may be at least partially reversed by treatment with aphytohormone.

Additionally, dramatic delay of flowering has been shown in othermonocots. In wheat, flowering was inhibited using agibberellin-degrading enzyme. This wheat, however, evidenced certaindeleterious side effects such as dwarfism when in the non-floweringphase. The present invention avoids these deleterious side effects.

Although inhibition of flowering in grasses is considered to be a traitof high agronomic value, we are unaware of any demonstration in theprior art that genetic modification of grass can result in anon-flowering phenotype. The present invention has a number ofsignificant advantages both for grasses bred for forage as well asgrasses bred for amenity purposes. These advantages can be summarised asfollows:

-   -   As a consequence of an extended vegetative growth phase, biomass        will be generated continuously in the form of leaf material,        which means a significant increase of the yield of        well-digestible organic matter.    -   The loss of nutritional quality of the crop as a consequence of        the formation of strongly lignified inflorescences as well as        seeds is prevented. The percentage of digestible organic matter        of a non-flowering grass is estimated to be about 80% during the        whole season whereas this percentage is estimated to be about        60% for a non-genetically modified flowering grass. This        reduction in nutritional value is prevented by the present        invention and the resulting increase in yield allows a farmer to        significantly lower the use of feed additives and thereby        minimise the overall emission of minerals into the environment.    -   Amenity grasses are improved in appearance and functional        properties due to increased tillering and the absence or        reduction of inflorescences.    -   Pollen development is blocked by a male-sterile phenotype such        as inhibition of flowering. Therefore, as an additional benefit        of the present invention, there is no production and spread of        pollen. The environment is protected thereby from the putative        risk of dissemination of traits conferred by transgenes (e.g.,        like herbicide resistance) to other plant species. Furthermore,        allergy sufferers are protected from aggravation of their        hayfever by this blockage.

Ectopic expression of AtH1, a gene encoding a homeotic transcriptionfactor involved in the pathway for phytochrome B signal transduction, inthe dicot plants Arabidopis and tobacco resulted in a delayed floweringphenotype. The phenotype could be reversed to flowering by exogenousapplication of gibberellic acid (see Intl. Patent Appln. No.PCT/IB98/00821 published as WO 98/51800).

In contrast, the mechanism that controls the transition to flowering ingrasses is currently unknown and persons skilled in the art had noreasonable expectation that the function of the AtH1 gene would beconserved in monocot species. Thus, the inhibition of flowering ingrasses and the switch from vegetative to generative growth, instead ofmere delay in flowering, was unexpected.

SUMMARY OF THE INVENTION

The present invention broadly encompasses a genetically modified grassin which generative propagation is inhibited or substantially reduced.Such inhibition is at least “substantial” in that there is a dramaticreduction in a phenotype (i.e., change in one or more plantcharacteristics resulting from the genetic modification) as compared tothe same species that has not been genetically modified. Morespecifically, it is directed to a non-flowering grass. The plant may bemale sterile or female sterile. Even more specifically, the geneticmodification may interfere with metabolism of gibberellic acid (e.g., byectopic expression of a homeobox gene encoding a transcription factor,in particular a transcription factor that blocks heading). Vegetativegrowth may be increased thereby. Thus, the digestibility and/ornutritional value of animal feedstuff may be improved.

Moreover, the present invention encompasses seed and other plant parts(e.g., pollen or ovum forming), at least some of which may be used forsexual or asexual propagation of the grass. The present invention may beused for forage or amenity purposes. Exemplary species useful for thepresent invention are of Dactylis glomerata L., Festuca arundinaceaschreb., Festuca pratensis huds., Lolium perenne L., Lolium multiflorumlam., Phleum pratense L., Agrostis tenuis sibth., Festuca rubra L.,Festuca ovina ssp. Duriuscula (L.) koch, Poa pratensis L., Poa trivialisL., Medicago saliva L., Trifolium pratense L., Trifolium repens L.,Agrostis L. Bermuda, Agrostis tenuis, and Agrostis stolonifera.

In addition, the present invention teaches methods of making and usingsuch genetically modified grasses. The genetic modification of the grassmay be produced by transformation of a grass species with a nucleicacid. For example, the nucleic acid may interfere with metabolism ofgibberellic acid. This nucleic acid can come from a monocot or dicot.The nucleic acid may express a gene encoding for a transcription factor(e.g., the homeobox gene AtH1 which can be derived from Arabidopsis orother equivalents thereof).

The phrase “ectoptic expression” is defined as expression of a gene at atime and/or in an amount that is different from the endogenous geneactivity and sufficient to confer the desired phenotype.

Optionally, the same or another nucleic acid may be introduced to conferanother linked or unlinked heritable trait (e.g., herbicide or pestresistance).

The genetically modified grass may be grown and/or propagated. It may beused for athletic fields, lawns, parks, and other types of landscaping(i.e., amenity uses). For example, sports such as baseball, cricket,football, golf, rugby, soccer, and tennis may be played on grass of thepresent invention. Animals such as livestock (e.g., cattle, goats,horses, sheep) may graze directly thereon or eat feed processed from thegenetically modified grass (i.e., forage uses). The invention provides amore digestible feedstuff for ruminant animals than the parentalflowering grass even after extensive cuttings.

The genetic modification may result in a heritable change in one or moreplant characteristics such as, for example, inhibition of flowering (orsubstantial delay that amounts to inhibition), absence of inflorescence,increased production of tillers, delayed heading, and inhibition of thedevelopmental switch from vegetative to generative growth.

It would be useful to be able to relieve or reverse one or more suchchanges. For example, expression of a gene may be normalized or aphytohormone may be applied to the grass to restore gibberellic acidmetabolism. The phytohormone may be a gibberellin compound in its acidor salt, ether or ester forms; it may be formulated with a carrier thatenhances penetration (e.g., dimethyl sulfoxide, alcohol, surfactant). Aswitch to generative propagation may be induced by genetic or chemicalmethods.

Another aspect of the invention is related to preventing the escapeand/or spread in the environment of one or more other plantcharacteristics (e.g., herbicide or pest resistance) that have also beengenetically engineered as a trait. Thus, the putative risk associatedwith the spread of genetically engineered traits to non-modified plantrelatives is minimised.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a physical map of pVDH309.

FIG. 2 shows a physical map of pVDH624.

FIG. 3 shows a physical map of pVDH633.

FIG. 4 shows a physical map of pVDH634.

FIG. 5A shows a physical map of pVDH410.

FIG. 5B shows a physical map of pVDH608.

FIG. 5C shows a physical map of pVDH619.

FIG. 5D shows a physical map of pVDH632.

FIG. 6 shows a physical map of pVDH636, its nucleotide sequence (SEQ IDNO:1), the molecular features of pVDH636, and the predicted amino acidsequence (SEQ ID NO:2) of AtH1.

FIG. 7 shows an ethidium bromide-stained agarose gel in which bands wereobtained after PCR analysis of AtH1-transformants using genomic DNAextracted from leaves. On the left-hand side of each panel (i.e., lanes1, 26, 51 and 76), a molecular size marker (lambda DNA digested withHindIII) is shown. Remaining lanes (i.e., lanes 2-25, 27-50, 52-75 and77-100) contain the PCR product obtained from independent transformants.Most samples analysed gave a positive PCR signal of the expected size of1463 basepairs (bp).

FIG. 8 shows a Southern analysis of independent AtH1-transformants ofLolium. Each lane contains HindIII digested genomic DNA isolated from adifferent, independent Lolium transformant. The blot was hybridisedusing labeled HPTII DNA as probe.

FIG. 9 shows an ethidium bromide-stained agarose gel in which bands wereobtained after RT-PCR analysis of AtH1-transformants using total RNAextracted from leaves. On the left-hand side of each panel (i.e., lanes1 and 8), a molecular size marker (lambda DNA digested with HindIII) isshown. Remaining lanes (lanes 2-7 and 9-14) contain the RT-PCR productobtained from independent transformants. The presence of a 1463 bp DNAfragment indicates the presence of a full-length AtH1 transcript in thetransformant.

FIG. 10 shows the phenotype of Lolium perenne transformants expressingAtH1. The upper panel shows the phenotype of an non-transformed plant(right) and an AtH1-transformant (left) three months after floweringinduction. The negative control plant shows an abundant number ofinflorescences whereas the AtH1 plant remains completely vegetative. Thelower panel shows the phenotype of the AtH1 transformant characterisedby a normal habitus and continued vegetative growth.

DESCRIPTION OF THE INVENTION

This invention describes a method to inhibit generative propagation ingrass. It can be used to block the transition to flowering in grasses,as well as to control the process to switch back to flowering when thisis desired.

This technology is useful in all grass species. They all have differentprimary uses, but the non-flowering technology is beneficial foragricultural use such as in alfalfa or in other forage grasses. Inamenity grasses, non-flowering increases visual uniformity of the grasstop and increases the lush bushiness of the lawn. Hardiness and ease ofmaintenance are plant characteristics desirable for areas that receiveheavy use such as, for example, publicly accessible areas like parks andathletic fields.

TABLE 1 Grasses for which the invention is particularly useful. Dactylisglomerata L. Cocksfoot Festuca arundinacea schreb. Tall fescue Festucapratensis huds. Meadow fescue Lolium perenne L. Perennial ryegrassLolium multiflorum lam. Italian ryegrass Lolium multiflorum lam.Westerwold ryegrass Phleum pratense L. Timothy Agrostis tenuis sibth.Browntop Festuca rubra L. Chewings fescue Festuca rubra L. Slendercreeping red fescue Festuca rubra L. Creeping fescue Festuca ovina ssp.Duriuscula (L.) koch Hard fescue Poa pratensis L. Smooth-stalkedmeadowgrass Poa trivialis L. Rough-stalked meadowgrass Medicago sativaL. Lucerne Trifolium pratense L. Red clover Trifolium repens L. Whiteclover Agrostis L. Bermuda Bent grass Agrostis tenuis Browntop bentAgrostis stolonifera Creeping bent

The term “grass” as used herein refers to those listed in Table 1 andother monocots commonly considered grass but not including thosemonocots commonly considered cereals such as corn, rice, wheat, barley,and the like.

Turfgrass seed must be evaluated for the suitability of differentcultivars for various amenity uses. There are a number of institutesthat test grasses for various uses. Most of the sports-type uses requirehigh levels of wear tolerance and shoot density. The invention avoidsgenerative growth and, thus, only exhibits vegetative growth under usualconditions. This vegetative growth results in more shoot density andmore wear tolerance. The combination of a cultivar that has been bred tohave excellent levels of shoot density and wear tolerance with thisgenetic modification is very promising for recreational and sports uses.Of particular interest are certain cultivars that are presently marketedin the U.K. such as Master perennial ryegrass for soccer and rugbypitches and Amadeus (Lolium perenne) for cricket fields and lawns.

Grass cultivars are ranked according to different sets of criteria bythe Sports Turf Research Institute (STRI) in England. For winter pitchesthe criteria produced by the STRI is based on mean wear tolerance overlow and high fertiliser inputs, and shoot density. These characteristicsare important for sports pitches receiving intensive wear such as soccerand rugby pitches. The invention increases the shoot density and thusresults in a grass that is superior for sports uses.

The invention can be employed in a number of grass types allowing theeffect of enhanced vegetative growth and tillering to be used in anumber of applications. When employed in a finer leafed grass theinvention is highly useful for lawns, parks, general landscaping, andahtletic fields. Perennial ryegrass (Lolium perenne) is tested by STRIfor tolerance of close mowing, shoot density, fineness of leaf, slowregrowth (regular mowing), mean, cleanness of cut, short growth(infrequent mowing), freedom from red thread, summer greenness andwinter greenness. The invention is particularly useful when introducedinto cultivars such as Bellevue which already evidence traits such asenhanced shoot density, fineness of leaf, and tolerance to close mowing.

Cultivars of Chewings fescue and slender creeping red fescue aresuitable for use in very close mown turf (for example golf and bowlinggreen mown at 5 mm) and for more general uses such as lawns and golffairways. For general turf, the cultivars are looked at for shootdensity and tolerance to close mowing. Tolerance of close mowing andshoot density will be of most importance for ornamental lawns and veryclose mown turf such as golf and bowling greens. The invention willenhance shoot density of Chewings fescue and slender creeping redfescue, and render them suitable for this type of use.

Cultivars of browntop bent (Agrostis tenuis) and creeping bent (Agrostisstolonifera) are used in golf and bowling greens which are closely mown,and for ornamental lawns and golf fairways. Velvet bentgrass is a denseturf and exhibits some drought tolerance. However, it also produces morethatch than other bentgrass species. When genetically modified toinhibit generative propagation and optionally to be herbicide resistant,grass with vegetative-only growth would enhance its useful for greensand fairways. Additionally, it may reduce mowing costs associated withremoval of seed heads of the grass. Thus, grasses of the Invention mayalso be aesthetically more pleasing to the eye than grasses which haveflowered.

Smooth-stalked meadowgrass (Poa pratensis) for use in winter pitches(soccer, rugby, etc.) is tested by STRI for wear tolerance, shootdensity, fineness of leaf, slow regrowth (regular mowing), freedom fromleaf spot, orange stripe rust resistance, summer greenness and wintergreenness. Such plant characteristics are clearly of importance foramenity uses. Therefore, the invention can be used for landscaping andsports either in combination with the naturally high levels of shootdensity or to increase the shoot density of cultivars lacking such atrait.

Cultivars of smooth-stalked meadowgrass (Poa pratensis) can be employedunder football-type wear for inclusion in winter pitches and forlandscaping (e.g., lawns and parks). Smooth-stalked meadowgrass istested by STRI on tolerance of shoot density, fineness of leaf, mean,slow regrowth (regular mowing), short growth (infrequent mowing),freedom from leaf spot, orange stripe rust resistance, summer greenness,and winter greenness. Once established, smooth-stalked meadowgrass canbe useful for football (soccer) wear and has tolerance of close mowing.However, establishment of this grass is slow and results cannot beachieved until at least 12 months after sowing. The invention mayenhance the establishment of the grass and reduce the down time prior touse.

U.S. golf courses frequently employ Agrostis L. (bent grasses) whilegolf courses in Europe employ the fine tillered meadow fecsue. Thesoccer fields of Europe frequently employ mixtures of Lolium perenne andPoa grasses. All of these grass uses are improved by the use of anon-flowering grass. Non-flowering grass increases the longevity underwearing conditions of the grass due to the bushiness resulting from theplant placing energy into the production of tillers instead of theproduction of inflorescence. Additionally, these additional tillerscreate a more level cut grass surface, (a uniform sward) which mayenhance ball directional control in golf for example. The increasedvegetative growth should reduce brown spots due to cleat or divotdamage. Additionally, the use of a non-flowering herbicide resistancegrass is particularly useful to decrease care and maintenance costsassociated with the removal of weeds from greens, pitches and fairwaysand roughs.

The invention can be introduced into other plants by conventionalbreeding methods such as forming hybrids, conventional breeding,backcrossing, or cross pollination, (after the non-flowering is switchedoff by application of a phytohormone which induces flowering). Oralternatively, the invention can be introduced into a grass by geneticmodification of the plant. This transition to non-flowering can bethrough introduction of genetic material (e.g., a nucleic acid like anexpression vector) by transformation processes.

Transition to flowering in a plant is a critical and complexdevelopmental process during the life cycle of a plant. The process iscontrolled by external factors like day length, light quality andquantity, low temperature, availability of water and nutrients.Moreover, internal factors like plant size, and number of internodes areconsidered to be critical. The plant senses this complex array ofenvironmental cues and this information is relayed to the nucleus wherethe gene expression profile is modulated in order to respondappropriately to the existing conditions. This mechanism maximises thechances of a plant to successfully produce viable offspring and thereforcontributes to its fitness.

These properties, which increase the survival rate in nature of theplant species, can be in conflict with the characteristics desired foragricultural use. Transition to flowering in grasses is a trait, whichlowers the benefits of this crop-group for agricultural use. However,for seed production, one of the objectives of grass breeding is toselect for varieties which are good flowering. However now that there isthe present invention an objective of grass breeding can includeselection of varieties which are delayed or completely blocked in theswitch to flowering. A prerequisite for grass seed production (but notfor sod production) is to design a controlled switch mechanism whichallows seed production when required.

The regulation of the flowering induction process is under control of alarge number of gene loci. Molecular genetic studies on Arabidopsiscurrently have identified a total number of 80 loci involved in thecontrol of flowering time. This complexity combined with the diversitywhich exist between plant species with respect to the floweringinduction process make it hard to predict which gene products are keyregulatory factors in the signal transduction cascades that control thetransition to flowering. As a consequence, the efficacy of exploitingavailable genetic factors in either homologous or heterologous systemswith the objective to control the developmental regulation cannot bepredicted on theoretical grounds but needs to be tested experimentallycase by case.

In WO 98/51800, the use has been described of a homeotic gene calledAtH1 derived from the dicotyledonous plant species Arabidopsis thalianato control the flowering induction process. Ectopic overexpression ofthe Ath1 gene in dicots like Arabidopsis and tobacco significantlyinhibited the transition to flowering, whereas downregulation of thisgene in Arabidopsis resulted in precocious flowering. Biochemicalanalysis showed that overexpression of the AtH1 gene in tobacco lowersthe endogenous concentration of biological active forms of thephytohormone gibberellic acid. The inhibited flowering phenotype can bereversed by exogenous application of gibberellic acid:

A number of formulations containing a gibberellin compound in variousforms such as ethers, esters, salts or acids could be used to induceflowering. Exemplary compounds are GA3 or 16,17-dihydro-GA3 or 3-epi-GA3or 3-epi-16,17-dihydro-GA3 or 2,2-dimethyl-GA4 and its 3α-OH derivativeand its 16,17-dihydro derivative and 3-epi-2,2-dimethyl-GA4 and its16,17-dihydro derivative and GA5 and its 16,17-dihydro derivative and15β-OH-GA5 and its 16,17-dihydro derivative including exo-16,17-dihydro-GA₅. Other examples include exo-16,17-dihydro-GA5,endo-16,17-dihydro-GA5, exo-16,17-dihydro-GA5-13-acetate,endo-16,17-dihydro-GA5-13-acetate, exo-16,17-dihydro-GA5-13-n-propylether.

The invention describes the use of the AtH1 gene in monocotyledonousplant species like grasses with the objective to control the floweringinduction process. Transition to flowering in grasses is characterisedby a three-month vernalisation requirement and consecutive long dayconditions. This differs from the Arabidopsis and tobacco varieties usedearlier to demonstrate the efficacy of AtH1 to control floweringinduction which do not require vernalisation and are day-lengthindependent. Therefor it could not be predicted and it was surprisingthat ectopic expression of AtH1 in transgenic grasses would result in adelayed and/or non-flowering phenotype. The tillering evidenced by thisvegetative growth in the plant and the lack of any negative phenotypechanges such as dwarfism or other abnormalities was very surprising.

Initially it was believed that a monocot homologue of the AtH1 genewould have to be found. It is still believed that this would be ausefully homologue as would most of the other monocots homologues fromcorn, lilies, rice, wheat and the like. But just as a inexpensive test,which was not expected to work, a DNA construct using the dicot gene wasmade. It was not evident that this dicot gene would be useful in amonocot. However, upon expression in of the dicot AtH1 gene thetransgenic grass plants showed an accumulation of the AtH1 protein whichthen might inhibit the flowering induction process.

The vector used to transform grass is based on pBluescript and containsthe AtH1 cDNA under transcriptional control of the ubiquitin promoterderived from maize. This promoter is constitutively active inmonocotyledonous species including Grass and is therefor useful tooverexpress transgenes. Additionally, it is noted that promoters thatare triggered to stop in the presence of the gibberellic acid are veryuseful. However, other promoters can be useful in this respect as well.Possibly tissue-specific promoters like promoters exclusively active inshoot apical meristem could also be used.

In order to select for transformants use is made of a HPTII gene, whichupon expression confers resistance towards the antibiotic hygromycin.Hygromycin has been shown to be very effective as a selective agent butother selectable marker systems could be used as well like kanamycinresistance, glyphosate resistance, gluphosinate-ammonium resistance, andthe like. In order to generate transgenic Lolium plants embryogenicsuspension cultures were bombarded using the so-called particle inflowgun (PIG). Other transformation systems can be used as well like thewhisker system or Agrobacterium tumefaciens. The transformationexperiments have resulted in a large number of hygromycin resistant,transgenic Lolium plants, which were characterized molecularly. Thenumber of integrated copies of the AtH1 construct was variable andranged from one to ten.

The transformants were analysed further by RT-PCR in order to selectthose transformants that express the integrated AtH1 gene. AtH1 mRNAcould be detected in about 70% of the transformants. A group of controlnon-transformed plants as well as the transgenic plants were used in aflowering experiment. In order to do this the plants were vernalised for10 weeks at an average temperature of 4° C. After the vernalisationperiod, the plants were placed under conditions favoring induction offlowering (i.e., long days of 16 hr light/8 hr dark) and 20° C. Plantswere monitored weekly for the appearance of inflorescences. Controlnon-transformed plants, which share the same genetic background as thetransformant plants, were developing inflorescences about three to sixweeks after transfer to long day conditions (99% of the individualplants). However, a significant number (i.e., 18%) of AtH1 expressing,independent transformants did not flower at all even at four monthsafter transfer to long days. This result shows that, surprisingly,ectopic expression of the Ath1 gene in transgenic grass can result in acomplete block of the transition to flowering. Moreover, thenon-flowering transformants continued developing vegetatively whichresulted in a large increase of biomass. No obvious negative pleiotropiceffects were observed for the non-flowering transformants.

In agronomic practice, fields are treated with phytotoxic, chemicalcompounds to control weeds. If these compounds are applied during thelife cycle of the crop, the crop needs to be resistant to the compound.One way to confer resistance to the otherwise susceptible crop is totransgenically introduce a resistance gene into the crop. In order tohave a sustainable system of susceptible weeds and a tolerant crop it ispertinent that the resistance gene does not flow into the germplasm ofthe weedy relatives of the crop. This is especially an issue incultivated grass crops, which have many wild relatives with whichgenetic material can be exchanged. A strategy to prevent the flow ofgenetic resistance traits from a cultivated crop into its wild and weedyrelatives is disclosed: a non-flowering genetic trait is combined with agene conferring resistance to the phytotoxic compound. Although not usedas such in agronomic practice, hygromycin is a phytotoxic compound.Grass plants treated with hygromycin in vitro die, unless they expressthe HPTII resistance gene.

The invention demonstrates that plants inhibited for generativepropagation as well as containing a transgene conferring resistance to aphytotoxic compound like hygromycin survive treatment with thephytotoxic compound, whereas control plants not expressing the transgeneconferring hygromycin resistance do not. This demonstrates that agenetic trait linked to a non-flowering gene can be used in vivo andthat the non-flowering technology is useful to lower the risk of thespreading of transgenes in the environment.

A normally flowering grass can be genetically modified by atransformation method such as a gun apparatus, an inflow apparatus(PIG), or an Agrobacterium which is adapted for monocot use. Thetransformation method must be capable of the introduction of afunctional gene construct. This gene construct should lead to thebiosynthesis and accumulation of a homeotic protein. The specifichomeotic protein or functional homologues (a functional homologueprotein is a protein that results in a novel and unexpected life cycleof the grass plant characterized by an extended vegetative growth phaseand inhibited generative growth phase) of that protein AtH1 originatingfrom the cruciferous plant species Arabidopsis thaliana results in anovel and unexpected life cycle of the grass plant characterized by anextended vegetative-growth phase and as a consequence a significantincrease in yield of biomass. This biomass is containing substantiallymore digestible feedstuff for ruminant animals than the floweringcontrol grass even after extensive numbers of grass cuttings.

Plants made in accordance with the invention were demonstrated tocontinue developing in a vegetative mode despite their being subjectedto environmental conditions strongly favoring the phase transition toflowering for non-transformed control plants having the same geneticbackground. A plant characteristic conferred by the invention can be atleast partially relieved or reversed by application of a phytohormone(e.g., a gibberellin compound).

The invention is further described by the following examples, but itspractice is not limited thereby.

EXAMPLES Example 1 Preparation of Transformation Vectors

In order to obtain transgenic grasses expressing the AtH1 gene derivedfrom A. thaliana (Quaedvlieg et al., 1995), an expression vector wasmade which contains the AtH1 cDNA under the transcriptional control of apromoter derived from the ubiquitin (UBI) gene from maize (Christensenet al., 1992), including the first exon-intron combination in order toenhance expression. The polyadenylation signal derived from the nopalinesynthase gene (Tnos) of Agrobacterium tumefaciens was attached at the3′-end of the cDNA to allow proper termination of transcription.Covalently linked to the chimeric AtH1 gene was a selectable markercomprised of the actin promoter (ACT) derived from rice, the HPTII genederived from Escherichia coli, and the 35S polyadenylation signal (T35S)derived from Cauliflower Mosaic Virus (McElroy et al., 1991; Spangenberget al., 1995a). Expression of the selectable marker confers-resistanceto the antibiotic hygromycin, which can be used to select transformedplants.

The construct was made using standard molecular cloning techniques andprotocols well known to the person skilled in the art. In detail theconstruct was made according to the following steps. The SacI site ofthe plasmid pVDH309 (FIG. 1), containing the UBI-promoter linked to agene encoding beta-glucuronidase (GUS), was made blunt by T4 DNApolymerase after which a NotI linker was attached to it. The resultingplasmid, called pVDH527, was digested with BamHI and NotI which removedthe GUS-gene which was subsequently replaced by a full length AtH1 cDNAwith a Tnos attached to the 3′-end which was released from plasmidpVDH619 (FIG. 5C) after digestion with Bg/II and NotI. The resultingplasmid is called pVDH624 (FIG. 2).

The primary structure of the UBI-AtHI construct was analysed bysequencing, which revealed a frame-shift mutation in the open readingframe of the AtH1 gene. To repair this mutation, a novel AtH1 cDNA wasprepared by PCR using a plasmid called pVDH608 (FIG. 5B) as template,which contained a correct version of the AtH1 cDNA. As the forwardprimer 5′-GCG TCG ACC CM TGG ACA ACA ACA ACA ACA AC-3′ (SEQ ID NO:3) andas the reverse primer 5′-GCG GAT CCG AGT AGC MT TGC CTA ATT ATC AC-3′(SEQ ID NO:4) were used. The PCR-product was digested with Sa/I andBamHI to generate sticky ends and ligated into pVDH624 digested withSa/I and BamHI, which resulted in plasmid pVDH632 (FIG. 5D). As theUBI-promoter also contains a Sa/I site, the Sa/I fragment of theUBI-promoter had to be introduced into pVDH632 in order to obtain theappropriate UBI-AtHI construct called pVDH633 (FIG. 3).

The XbaI site locate at the 5′-end of the UBI-promoter was modified to aNotI site by filling in the digested XbaI site with Klenow polymeraseand ligating a NotI linker to the blunt end. The resulting plasmid iscalled pVDH634, which is shown in FIG. 4. Sequence analysis showed thisUBI-AtHI construct to be correct. The plasmid pVDH634 was subsequentlydigested with NotI, which released the complete UBI-AtHI-NOS chimericgene. This DNA fragment was inserted into the NotI site of the plasmidpVDH410 (FIG. 5) which is a pUC-derived plasmid containing the ACT-HPTIIselectable marker with a unique NotI site at its 5′-end. The resultingvector, which contained both genes in the same transcriptionalorientation, was called pVDH636 (FIG. 6) and was used in Loliumtransformation experiments using the particle inflow gun as DNA deliverysystem. The integrity of the plasmid was confirmed by sequence analysis.The complete primary structure of pVDH636 is shown in FIG. 6. Inaddition, transformation was carried out using a mixture of the vectorspVDH410 and pVDH633.

Example 2 Transformation of Lolium perenne

Embryogenic suspension cultures of Lolium perenne L. (cv. Mondial) wereestablished (Spangenberg et al., 1995a) and transformed with pVDH636,using the particle inflow gun (PIG) (Finer et al., 1992). Filters withan embryogenic suspension culture were bombarded with gold particlescoated with the transformation vectors. Transformed tissues wereselected using hygromycin B according to Spangenberg et al. (1995b). Theresults of the transformation using pVDH636 are shown below.

TABLE 2 Summary of Lolium transformations using pVDH636 Minimum # of #filters # filters with independent Plasmid bombarded hyg^(R) shoots #transformants transformants pVDH636 787 306 943 279

As can be seen from the results shown in Table 2, approximately 39% ofthe filters carrying the embryogenic suspension cultures ultimatelyresulted in hygromycin resistant shoots. After transfer to rootingmedium, a total number of 943 putative transformants were obtained.However, as individual plants which are derived from one and the samefilter are considered to be possibly dependent (i.e. geneticallyidentical), the total number of independent transformants as defined asthe number of hygromycin resistant plants regenerated from differentfilters was 279.

In a separate transformation experiment co-bombardment of a mixture oftwo transformation vectors was carried out. This allows geneticsegregation between the integrated ACT-HPTII construct and theintegrated UBI-AtH1 construct in offspring for those events in which thetwo integrated plasmids are not genetically linked. The two vectors usedfor this transformation experiment were pVDH410, which contains theACT-HPTII selectable marker, and pVDH633, which contains the UBI-AtH1construct. The results of the transformation using pVDH410 and pVDH636are shown below.

TABLE 3 Summary of Lolium transformations using pVDH410 and pVDH636 #filters Minimum # of # filters with hyg^(R) # independent Plasmidsbombarded shoots transformants transformants pVDH410 + 257 107 129 67pVDH633

As can be seen from the results shown in Table 3, approximately 42% ofthe filters carrying the embryogenic suspension cultures ultimatelyresulted in hygromycin resistant shoots. After transfer to rootingmedium, a total number of 129 putative transformants were obtained.However, as individual plants which are derived from one and the samefilter are considered to be possibly dependent (i.e., geneticallyidentical), the total number of independent transformants as defined asthe number of hygromycin resistant plants regenerated from differentfilters was 67.

Example 3 Molecular Analysis of the Putative Transformants

In order to select transformants, which contain a functional UBI-AtH1construct, the hygromycin resistant plants were analysed molecularly. Aninitial screen was carried out by PCR to select for plants containing aminimum of one full-length copy of the AtH1 cDNA. Genomic DNA waspurified from leaf explants and used in a PCR reaction containing thefollowing primer set: forward primer 5′-GCG TCG ACC CM TGG ACA ACA ACAACA ACA AC-3′ (SEQ ID NO:3) and reverse primer 5′-GCG GAT CCG AGT AGC MTTGC CTA ATT ATC AC-3′ (SEQ ID NO:4). The 1463 kb DNA fragment diagnosticfor the presence of an integrated full length AtH cDNA was observed in85% of the independent hygromycin resistant plants (FIG. 7). An estimateof the integrated number of gene copies was made by Southern analysisusing the restriction enzyme HindIII to digest the genomic DNA and HPTIIas a labeled probe. The result of such an analysis of plants transformedwith pVDH636 is given in FIG. 8 and shows from the different bandingpatterns that a number of independent events have been obtained whichcontain estimated copy numbers ranging from one to ten.

The PCR positive transformants identified above were further analysedfor the presence of full-length AtH1 mRNA in an RT-PCR reaction usingtotal leaf RNA as a template and 5′-GCG TCG ACC CM TGG ACA ACA ACA ACAACA AC-3′ (SEQ ID NO:3) as forward primer and 5′-GCG GAT CCG AGT AGC MTTGC CTA ATT ATC AC-3′ (SEQ ID NO:4) as reverse primer. A positive signalwas obtained for more than about 70% of the transformants indicatingthat these transformants accumulate full-length AtH1 mRNA (FIG. 9).

Example 4 Phenotypic Analysis of Grass Transformants Expressing the AtH1Gene Derived from Arabidopsis thaliana

RT-PCR positive plants were vernalised (70 days at 4° C.), together withcontrol plants (RT-PCR negative plants, PCR negative plants, andnon-transformed plants). While control plants formed large numbers ofinflorescences under Long Day (LD) conditions in the greenhouse (3-6weeks after transfer from vernalisation to LD conditions), severalRT-PCR positive plants continued to form leaves, became very leafy, andhad not formed inflorescences 4 months after transfer to LD conditions(FIG. 10). Some RT-PCR positive plants formed only one or twoinflorescences about four months after transfer to LD conditions. Theoverall result is given in Table 4.

TABLE 4 Summary of flowering experiment using AtH1 expressers of LoliumRT-PCR Non-transformed positive plants control plants Total # of plants185 101 # of non-flowering plants 34 1

Example 5 Transformation of Poa pratensis L.

Embryogenic suspension cultures of Poa pratensis L. (Kentucky bluegrass)(cv. Geronimo) are established according to Nielsen and Knudsen (1998).Genetic transformation for suspension cultures are carried out asdescribed by Spangenberg et al. (1995b) and transformation is withpVDH636, using the particle inflow gun (PIG). The tissue which istransformed is selected, using hygromycin B, according to Spangenberg etal. (1995b).

Example 6 Transformation of Festuca arundinacea and Festuca rubra

Embryogenic suspension cultures of tall fescue (Festuca arundinaceaSchreb.) or red fescue (Festuca rubra L.) are established, and are thensubjected to genetic transformation with pVDH636 according toSpangenberg et al. (1995b). Filters with an embryogenic suspensionculture are subjected to bombardment with gold particles coated with thetransformation vectors. Transformed tissues can be selected usinghygromycin B in accordance with Spangenberg et al. (1995b).

Example 7 Herbicide Resistance as a Trait Linked to a Gene InhibitingInduction of Flowering in Grasses

Transformants exhibiting a clear non-flowering phenotype (i.e.,substantial inhibition of flowering under vernalising conditions) wereused to demonstrate the functionality in vivo of a transgenic traitincreasing chemical resistance of a grass when this trait is geneticallylinked to the genetic modification. A control group of non-transformedLolium plants as well as a group of clonally propagated transgenicLolium plants transformed with pVDH636 and inhibited in flowering wereexposed to a phytotoxic compound through foliar application solution ofhygromycin B. The control plants showed severe damage as a consequenceof the treatment with hygromycin B. However, the non-floweringtransformants are able to survive this treatment as a consequence of thepresence of a genetically linked functional hygromycin resistance gene.

Example 8 Genetic Transformation of Grass for Both Inhibition ofGenerative Propagation and Herbicide Resistance

Embryogenic suspension cultures of perennial ryegrass, tall fescue, andred fescue are established as described above, and are geneticallytransformed, using mixtures of plasmid pVDH636 and pUBA (Toki et al.,1992). Selection of transformed tissues is carried out using hygromycinas described. Non-flowering plants are sprayed with a 1% (v/v) Basta(glufosinate) solution containing 0.1% (v/v) Tween 20 (Toki et al.,1992), in order to detect plants that are both non-flowering andherbicide-resistant.

The usefulness of a non-flowering herbicide-resistant grass is selfevident. It avoids the issues of the herbicide resistant being spread toother species of grass or to weeds. The invention can be made with aglufosinate-resistant gene such as Pat or Bar (see EP 0257542 and EP0275957), a glyphosate gene such as the monocot gene (see U.S. Pat. No.5,554,798), or a gene of the EPSP class (see U.S. Pat. No. 4,940,835).Additionally, resistance to herbicides containing imidazolinones (e.g.,Pursuit), can be introduced with the gene encoding a mutant AHAS enzyme(see U.S. Pat. No. 5,731,180). Furthermore, the combination of (1) aknown gene that confers herbicide or pest resistance and (2) a geneticmodification which inhibits generative propagation is also envisioned.

Example 9 Reversing Non-Flowering in Grass with Gibberellin

All independent transformed plants, expressing the ATH1-gene, werecloned to form two sets of plants. All plants were vernalized (16 hrdark/8 hr light at 4° C. for 70 days). After the vernalization periodthe plants were subjected to Long Day conditions (16 hr light/8 hr darkat 18° C.). One set of all transgenics was treated with gibberellic acid(GA3) (3×10⁻⁵ M GA3 in 5% ethanol by spraying) weekly for four weeks,starting at the start of the LD-period. The other set of plants wassprayed with 5% ethanol. At least some GA3-treated plants are expectedto form inflorescences, while the untreated counterparts of the sameevent will not form inflorescences. This result will show that GA3 canswitch the ATH1-induced inhibition of flowering in L. perenne to thenormal flowering mode. This reversal of phenotype may be enhanced bychemical penetration agents (e.g., DSMO, ethanol, surfactants) or byexposing the meristem to gibberellin by trimming away tillers and othervegetative growth.

Example 10 Relieving Delayed Heading in Grass with Gibberellin

Primary transformants derived from Lolium perenne L. (perennialryegrass) using pVDH636 were vernalized and then subjected to Long Day(LD) conditions in the greenhouse (17 hr light/7 hr dark). ManyAtH1-expressing transformants (i.e., RT-PCR positive) showed delayedheading, an important plant characteristic of grass, as compared tonon-expressing transformant or non-transformant controls. Several plantsfailed to flower three months after transfer to LD conditions.AtH1-expressing plants are generally very leafy (FIG. 10).

Several AtH1-expressing transformants (15-20 clones per transformant,three replications), which showed the delayed heading phenotypeconferred by the AtH1 transgene, were clonally propagated, vernalised inthe winter, and then subjected to a second round of LD conditions thefollowing spring.

Four different gibberellin compounds, which differ in theirflorigenicity (Evans et al., 1990 were used to treat 3-4 clones percompound and their effect on heading time of transformants and controlswas observed. Gibberellin was applied in solvent (5% ethanolsupplemented with 0.01% Tween-20 surfactant) at 30 mg/L. It was appliedby spraying six times with about 2-3 ml per plant over two weeks,started one week after the beginning of LD conditions. Mean heading timeis shown in days after the first spraying. Non-transformant controlswere treated with the solvent only.

Transformant (T) and non-transformant (NT) control plants (Table 5) weretreated with the indicated gibberellin. GA5 significantly stimulatedheading, and GA20 somewhat delayed heading, as compared to non-treatedcontrols. Variation between transformants for their sensitivity togibberellins could be great.

TABLE 5 Relief of Delayed Heading by Gibberellins (GA) Treatment MeanHeading Time T-GA5 33.5^(a) T-GA3 34.1^(ab) T-diHGA5 34.2^(ab) T-GA2037.8^(b) T-no GA 37.7^(b) NT-no GA 29.9^(a) Different superscriptedletters indicate statistically significant differences in mean headingtime (= 0.05 ANOVA).

Transformant (T) plants were grouped into PCR positive (+) or PCRnegative (−) for all gibberellin treatments and compared tonon-transformant (NT) control plants (Table 6). Only transformantsharbouring the AtH1 transgene on average head later than those lackingthe gene. Differences between transformants were great. Mean headingtimes ranged from about 27 days to about 59 days after the firstspraying.

TABLE 6 Relief Requires the Presence of the AtH1 Transgene TreatmentMean Heading Time T-PCR(+) 37.3^(b) T-PCR(−) 33.6^(a) NT-no GA 29.9^(a)Different superscripted letters indicate statistically significantdifferences in mean heading time (= 0.05 ANOVA).

Transformant plants were grouped into Taqman assay positive or PCR (+)or Taqman assay negative or PCR (−), and compared to non-transformantcontrol plants (Table 7). This allows differences to be seen betweentransformants that transcribe or do not transcribe the AtH1 transgene.AtH1-expressing transformants on average head later than those notexpressing the transgene. Tissue culture and particle bombardment canalso cause delayed heading in Lolium perenne (Stadelmann et al., 1998).

TABLE 7 Relief Requires Expression of the AtH1 Transgene Treatment MeanHeading Time PCR (+) expressing 48.7^(c) PCR (−) 42.6^(b) NT 32.4^(a)Different superscripted letters indicate statistically significantdifferences in mean heading time (= 0.05 ANOVA).

REFERENCES

-   Christensen et al. (1992) Plant Mol. Biol. 18, 675-689-   Evans et al. (1990) Planta 182, 97-106-   Finer et al. (1992) Plant Cell Reports 11, 323-328-   McElroy et al. (1991) Mol. Gen. Genetics 231, 150-160-   Nielsen and Knudsen (1998) J. Plant Physiol. 141, 589-595-   Quaedviieg et al. (1995) Plant Cell 7, 117-129-   Spangenberg et al. (1995a) Plant Science 108, 209-217-   Spangenberg et al. (1995b) J. Plant Physiol. 145, 693-701-   Stadelmann et al. (1998) Theor. Appl. Genet. 96, 634-639-   Toki et al. (1992) Plant Physiol. 100, 1503-1507

All publications cited herein are incorporated by reference and indicatethe level of skill in the art.

While the invention has been described in connection with what ispresently considered to be practical and preferred embodiments, itshould be understood that it is not to be limited or restricted to thedisclosed embodiments but, on the contrary, is intended to cover variousmodifications, substitutions, and combinations within the scope of theappended claims. In this respect, it should be noted that the protectionconferred by the claims is determined after their issuance in view oflater technical developments and would extend to all legal equivalents.

Therefore, it is to be understood that variations in the invention thatare not described herein will be obvious to a person skilled in the artand could be practiced without departing from the invention's novel andnon-obvious elements with the proviso that the prior art is excluded.

1. A grass which has been genetically modified by introducing a nucleicacid molecule encoding the homeobox transcription factor AtH1 operablylinked to a constitutive promoter, wherein said modified grass shows aninhibition of flowering as compared with a grass that has not beengenetically modified.
 2. The grass according to claim 1, wherein saidgenetic modification improves at least digestibility or nutritionalvalue or both of the grass with respect to a non-genetically modifiedplant of the same species.
 3. The grass according to claim 1, whereinsaid genetic modification results in a change in one or more plantcharacteristics selected from the group consisting of absence ofinflorescences, increase in production of tillers, and delay in heading,as compared with a non-genetically modified plant of the same species.4. The grass according to claim 1, wherein said genetic modificationresults in an increase in vegetative growth relative to non-geneticallymodified grass.
 5. The grass according to claim 1, wherein the geneticmodification lowers the endogenous concentration of gibberellic acid ascompared with a non-genetically modified plant of the same species. 6.The grass according to claim 1 which is an amenity-type grass.
 7. Thegrass according to claim 1 which is a forage-type grass.
 8. The grassaccording to claim 7 wherein said grass is a plant species selected fromthe group consisting of Dactylis glomerata L., Festuca arundinaccaschreb., Festuca pratensis huds., Lolium perenne L., Loluim multiflorumlam., Phleum pratense L., Agrostis tenuis sibth., Festuca rubra L.,Festuca ovina ssp. Duriuscula (L.) koch, Poa pratensis L., Poa trivialisL., Medicago sativa L., Trifolium pratense L., Trifolium repens L.,Agrostis L. Bermuda, Agrostis tenais, and Agrostis stolonifera.
 9. Theprogeny of the grass according to claim 1, wherein the progeny stablyinherited the genetic modification that substantially inhibitedgenerative propagation.
 10. A plant part of the grass according to claim1, wherein the plant part stably inherited the genetic modification thatsubstantially inhibited generative propagation.
 11. The plant partaccording to claim 9 wherein said plant part is a seed.
 12. A method ofmaking the grass according to claim 1 comprising transforming a grasswith a nucleic acid molecule that encodes the homeobox transcriptionfactor AtH1 operably linked to a constitutive promoter.
 13. A method ofusing the grass according to claim 1 comprising at least growth orpropagation or both of the grass.
 14. The method according to claim 13,wherein the grass is used to play at least one sport selected from thegroup consisting of baseball, cricket, football, golf, rugby, soccer,and tennis.
 15. The method according to claim 13, wherein the grass isused at least in a portion of an athletic field, lawn, or park.
 16. Themethod according to claim 13, wherein the grass is fed to an animalselected from the group consisting of a cow, a goat, a horse, and asheep.
 17. The method according to claim 13, wherein the grass is usedas animal feedstuff.